The cell resealing technique for manipulating, visualizing, and elucidating molecular functions in living cells

Background: Cell-based assays are essential for analyzing molecular functions and spatiotemporal information. The cell resealing technique, in which pore-forming toxins are used to permeabilize cell membranes, enables the delivery of various membrane-impermeable molecules inside cells. Scope of review: We review the basics of the resealed cell system, including optimized protocols, assessment of cellular damage, and recovery following permeabilization of the membrane. Additionally, we introduce the streptolysin O (SLO)-type and listeriolysin O (LLO)-type resealing techniques. In SLO, the formation of larger pores (similar to 30 nm) enables the passage of a wider range of molecules. Then, we discuss the advantages and applications of the semi-intact cell system, in which ongoing permeabilization is selected to maintain and analyze a specific cellular environment. Major conclusions: As confirmed by the effective use of quantitative image analysis, the SLO-type resealing system is successful for establishing and phenotyping diabetic model cells by introducing cytosol from diabetic mice. The LLO-type resealing technique enables the delivery of mid-sized molecules with high efficiency and low damage. As each technique has specific advantages, understanding the characteristics of LLO and SLO is necessary for choosing the appropriate technique. General significance: SLO-type resealing is optimal for creating disease model cells and drug screening, especially lifestyle-related diseases. LLO-type resealing is expected to be suitable for screening mid-sized biological drugs. Semi-intact cells can contribute to elucidating various cellular phenomena that have remained intractable due to their complexity.